1. Field of the Invention
This invention relates to the field of biochemical assays involving regulated expression of reporter genes, and to mutant strains of bacteria useful in biochemical assays. More particularly, it relates to methods of screening for molecules capable of affecting expression and/or activity of type III secretion machinery in gram-negative bacteria.
2. Description of Related Art
Type III secretion machinery is present in numerous gram-negative bacteria (including members of the species Shigella, Salmonella, Yersinia, Escherichia, Pseudomonas, Xanthomonas, Ralstonia, and Erwinia) that are pathogenic for man, animals, and plants. For example, the Sec-independent type III secretion pathway is involved in secretion of Yersinia anti-host proteins. In Salmonella and Shigella species, it is involved in the process of entry into epithelial cells. It is also implicated in EPEC signal transducing proteins, Pseudomonas aeruginosa toxins, and virulence factors of many plant pathogens, as well as in flagellum assembly of bacteria such as S. typhimurium and Bacillus subtilis.
Features of this secretion pathway can include activation of secretion by contact of the bacterium with host cells (Menard et al., 1994, "The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD.", EMBO J., 13:5293-5302; Watarai et al., 1995, "Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness.", EMBO J., 14:2461-2470; Zierler and Galan, 1995, "Contact with cultures epithelial cells stimulates secretion of Salmonella typhimurium invasion proteins InvJ.", Infect. Immun., 63:4024-4028); that some of the secreted proteins are delivered into the cytoplasm of host cells (Rosqvist et al., 1994, "Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells.", EMBO J., 13:964-972; Sory and Cornelis, 1994, "Translocation of an hybrid YopE-adenylate-cyclase from Yersinia enterocolitica into HeLa cells.", Mol. Microbiol., 14:583-594; Wood et al., 1996, "SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry.", Mol. Microbiol., 22:327-338; Collazo and Galan, 1997, "The invasion-associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell.", Mol. Microbiol, 24:747-756); and that transcription of genes encoding secreted proteins is controlled by secretion of regulatory proteins (Hughes et al., 1993, "Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator.", Science, 262:1277-1280; Pettersson et al., 1996, "Modulation of virulence factor expression by pathogen target cell contact.", Science, 273:1231-1233).
Based on the observations that (1) the secretion machinery is involved in secretion of factors which are active against the host, and (2) secretion mutants are avirulent, the type III secretion machinery provides an attractive target for the screening of molecules that would prevent or inhibit gram-negative bacteria from secreting their virulence factors. However, the search for molecules capable of inhibiting the secretion mechanism has previously required two conditions to be present. First, the type III secretion machinery must be active. And second, the product of the secretion activity, i.e., the secreted proteins, must be measurable. Unfortunately, the secretion machinery is, at best, only weakly active when bacteria are grown in standard laboratory media, making the search for inhibitor molecules difficult or impossible. In addition, there is no way to easily measure the presence of a protein secreted in the culture medium by the type III secretion machinery. These proteins do not have an easily assayable enzymatic activity and their secretion must be evaluated using ELISA, which is time consuming and expensive.